Dimer of the precursor of HIV-2 envelope glycoprotein

ABSTRACT

Four glycoproteins of apparent molecular weights 300,000, 140,000, 125,000, and 36,000 (gp300, gp140, gp125, and gp36) are detectable in human immunodeficiency virus type 2 (HIV-2) infected cells. The gp125 and gp36 are the external and transmembrane components, respectively, of the envelope glycoproteins of HIV-2 mature virions. The gp300, which is a dimeric form of gp140, the precursor of HIV-2 envelope glycoprotein, is probably formed by a pH dependent fusion in the endoplasmic reticulum. Such a doublet is also observed in cells infected with simian immunodeficiency virus (SIV), a virus closely related to HIV-2. On the other hand, the envelope glycoprotein precursor of HIV-1 does not form a dimer during its processing. Experiments carried out with various inhibitors of oligosaccharide trimming enzymes suggest that transient dimerization of the glycoprotein precursor is required for its efficient transport to the Golgi apparatus and for its processing. The gp300 is useful for detecting antibodies to HIV-2 antigens in human body fluids and for raising antibodies to gp300.

This application is a continuation of application Ser. No. 07/204,346filed Jun. 9, 1988.

BACKGROUND OF THE INVENTION

This invention relates to viral proteins and glycoproteins, tocompositions containing these proteins, to methods of preparing theproteins, and to their use in detecting viral infection.

The etiological agent of acquired immunodeficiency syndrome (AIDS) isthe retrovirus referred to as human immunodeficiency virus (HIV)(Montagnier et al., 1984). To date, two related but distinct viruses,HIV-1 and HIV-2, have been identified (Barre-Sinoussi et al., 1983;Popovic et al., 1984; Levy et al., 1984; Wain-Hobson et al., 1985a;Clavel et al., 1986a; Brun-Vezinet et al., 1987; Guyader et al., 1987).HIV-2 is closely related to simian immunodeficiency virus (SIV), whichcauses an AIDS-like disease in macaques (Daniel et al, 1985; Sonigo etal., 1985; Chakrabarti et al., 1987).

HIV-1, HIV-2, and SIV show all the features of retrovirus family members(Wain-Hobson et al., 1985b; Montagnier and Alizon, 1987; Guyader et al.,1987; Chakrabarti et al., 1987). Their proviral genomes contain two longterminal repeats (LTRs) and three essential genes required for virusreplication encoding the viral internal structural proteins (gag), thereverse transcriptase (pol), and the envelope glycoproteins (env) of thevirus. In addition to these genes, both HIVs and SIV contain additionalgenes encoding the proteins which regulate viral expression (tat andart/trs) and three other genes encoding proteins of unknown function (Qor sor, F or 3'orf, and R). The only notable difference in the geneticorganizations of HIV-1, HIV-2, and SIV resides in the open reading framereferred to as X, which is absent in HIV-1.

Alignments of the nucleotide sequences of HIV-1, HIV-2, and SIV reveal aconsiderable homology between HIV-2 and SIV. These two viruses shareabout 75% overall nucleotide sequence homology, but both of them areonly distantly related to HIV-1 with about 40% overall homology (Guyaderet al., 1987; Chakrabarti et al., 1987). At the protein level, the gagand pol proteins of HIV-1, HIV-2, and SIV are antigenicallycross-reactive, whereas env proteins are cross-reactive only betweenHIV-2 and SIV (Clavel et al., 1986b, 1987).

HIV-1, HIV-2, and SIV are both tropic and cytopathic for CD4 positive Tlymphocytes (Dagleish et al., 1984; Klatzman et al., 1984; McDougal etal., 1985; Clavel et al., 1986b, 1987; Kannagi et al., 1985; Fultz etal., 1986). A great number of studies have indicated that CD4 functionsas the cellular receptor for HIV-1 (for references see Weiss, 1988).

The HIV-1 env gene codes for a 160Kd glycoprotein that isproteolytically cleaved to yield the extracellular and transmembraneproteins, gp120 and gp41, respectively (Montagnier et al., 1985). It hasbeen demonstrated that HIV-1 recognition of CD4 is mediated by gp120.This complex gp120-CD4 can be identified by co-immunoprecipitation usingantibodies specific for the CD4 antigen (McDougal et al., 1986).Following the binding of gp120 to CD4, the entry of HIV-1 into the cellmight occur by viral envelope cell membrane fusion (Lifson et al., 1986;Sodroski et al., 1986; Stein et al., 1987; McClure et al., 1988). Aputative fusogenic domain in gp41 (Kowalski et al., 1987), which has asequence homologous to other fusion peptides (Phe-Leu-Gly; Gallaher,1987), might provide at least one HIV fusion site necessary for thisprocess (Marsh and Dalgleish, 1988).

In the case of HIV-2, a high molecular weight protein of about 130Kd toabout 140Kd has been associated with the major envelope glycoprotein(Clavel et al., Science, 233:343-346, 1986). Another glycoprotein havinga molecular weight of 120Kd has been associated with the externalglycoprotein of HIV-2 (Guyada et al., Nature, 362:662-669, 1987).Nevertheless, detailed information for HIV-2 envelope proteins andglycoproteins and their cleavage products and precursors is lacking.

There exists a need in the art for additional information on thestructure and in vivo processing of HIV-2 proteins, and especially HIV-2envelope proteins and glycoproteins. Such information would aid inidentifying HIV-2 infection in individuals. In addition, such findingscould aid in elucidating the mechanism by which HIV-2 infection andvirus proliferation occur and thereby make it possible to devise modesof intervening in viral processes.

SUMMARY OF THE INVENTION

This invention aids in fulfilling these needs in the art by providingHIV-2 envelope proteins and glycoproteins in purified form. Fourglycoproteins of apparent molecular weights 300,000, 140,000, 125,000,and 36,000 daltons (gp300, gp140, gp125 and gp36) are detectable inhuman immunodeficiency virus-2 (HIV-2) infected cells. The gp125 andgp36 are the external and transmembrane components, respectively, of theenvelope glycoproteins of HIV-2 mature virions. It has now beendiscovered that the gp300 is a dimeric form of gp140, which is theprecursor of HIV-2 envelope glycoprotein. This invention thus providesgp300 glycoprotein of HIV-2 and human retroviral variants of HIV-2 inpurified form.

This invention also provides proteins of HIV-2 or of a human retroviralvariant of HIV-2 having apparent molecular weights of about 200Kd (p200)and about 90 to about 80 Kd (p90/80). These proteins are substantiallyunglycosylated and are in a purified form.

A similar high molecular weight glycoprotein of Simian ImmunodeficiencyVirus (SIV) or of a Simian retroviral variant of SIV has also beendiscovered. This glycoprotein is a precursor of an envelope glycoproteinof SIV and has an apparent molecular weight of about 300Kd (gp300SIV).This glycoprotein is also provided in a purified form.

This invention also provides labeled gp300 of HIV-2 and gp300 of SIV.Preferably, the labeled glycoproteins are in purified form. It is alsopreferred that the labeled glycoprotein is capable of beingimmunologically recognized by human body fluid containing antibodies toHIV-2 or SIV. The gp300 glycoproteins can be labeled, for example, withan immunoassay label selected from the group consisting of radioactive,enzymatic, fluorescent, chemiluminescent labels, and chromophores.

Immunological complexes between the proteins and glycoproteins of theinvention and antibodies recognizing the proteins and glycoproteins arealso provided. The immunological complexes can be labeled with animmunoassay label selected from the group consisting of radioactive,enzymatic, fluorescent, chemiluminescent labels, and chromophores.

Furthermore, this invention provides a method for detecting infection ofcells by human immunodeficiency virus type-2 (HIV-2). The methodcomprises providing a composition comprising cells suspected of beinginfected with HIV-2, disrupting cells in the composition to exposeintracellular proteins, and assaying the exposed intracellular proteinsfor the presence of gp300 glycoprotein of HIV-2. The exposedintracellular proteins are typically assayed by electrophoresis or byimmunoassay with antibodies that are immunologically reactive with gp300glycoprotein of HIV-2.

This invention provides still another method of detecting antigens ofHIV-2, which comprises providing a composition suspected of containingantigens of HIV-2, and assaying the composition for the presence ofgp300 glycoprotein of HIV-2. The composition is typically free ofcellular debris.

A method of distinguishing HIV-2 infection from HIV-1 infection in cellssuspected of being infected therewith has also been discovered. Themethod comprises providing an extract containing intracellular proteinsof the cells, and assaying the extract for the presence of gp300glycoprotein. The gp300 is characteristic of HIV-2, but the glycoproteinhas not been found in extracts of HIV-1 cell cultures.

In addition, this invention provides a method of making gp300glycoprotein of HIV-2, which comprises providing a compositioncontaining cells in which HIV-2 is capable of replicating, infecting thecells with HIV-2, and culturing the cells under conditions to causeHIV-2 to proliferate. The cells are then disrupted to exposeintracellular proteins. The gp300 glycoprotein is recovered from theresulting exposed intracellular proteins.

This invention also provides an in vitro diagnostic method for thedetection of the presence or absence of antibodies which bind to anantigen comprising the proteins or glycoproteins of the invention ormixtures of the proteins and glycoproteins. The method comprisescontacting the antigen with a biological fluid for a time and underconditions sufficent for the antigen and antibodies in the biologicalfluid to form an antigen-antibody complex, and then detecting theformation of the complex. The detecting step can further comprisemeasuring the formation of the antigen-antibody complex. The formationof the antigen-antibody complex is preferably measured by immunoassaybased on Western Blot technique, ELISA (enzyme linked immunosorbentassay), indirect immunofluourescent assay, or immunoprecipitation assay.

A diagnostic kit for the detection of the presence or absence ofantibodies which bind to the proteins or glycoproteins of the inventionor mixtures of the proteins and glycoproteins contains antigencomprising the proteins, glycoproteins, or mixtures thereof and meansfor detecting the formation of immune complex between the antigen andantibodies. The antigens and the means are present in an amountsufficient to perform the detection.

Precursors of the envelope glycoproteins of HIV-2 and SIV can beprepared according to this invention. Specifically, this inventionprovides a method of preparing the precursors, which comprises providingan extracellular composition containing gp300 glycoprotein of HIV-2 orSIV at a pH of at least about 6.5. The pH of the composition is thenlowered to a value of about 4 to about 6.0 in order dissociate the gp300glycoprotein into gp140 glycoprotein of HIV-2 or gp140 glycoprotein ofSIV.

Finally, this invention provides an immunogenic composition comprising aprotein or glycoprotein of the invention in an amount sufficient toinduce an immunogenic response in vivo, in association with apharmaceutically acceptable carrier therefor.

The proteins and glycoprotein of this invention are thus useful as aportion of a diagnostic composition for detecting the presence ofantibodies to antigenic proteins associated with HIV-2 and SIV. Inaddition, the proteins and glycoproteins can be used to raise antibodiesfor detecting the presence of antigenic proteins associated with HIV-2and SIV. The proteins and glycoproteins of the invention can be alsoemployed to raise neutralizing antibodies that either inactivate thevirus, reduce the viability of the virus in vivo, or inhibit or preventviral replication. The ability to elicit virus-neutralizing antibodiesis especially important when the proteins and glycoproteins of theinvention are used in vaccinating compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will be described in greater detail by referring to thedrawings in which:

FIG. 1A is a fluorograph in which high molecular weight proteins ofHIV-1 and HIV-2 are compared after electrophoresis in a polyacrylamideSDS-gel;

FIG. 1B depicts the result of electrophoresis of HIV-2 glycoproteins inan acrylamide gel;

FIG. 2 depicts the result of two dimensional gel electrophoreticanalysis of HIV-2 glycoproteins;

FIG. 3(a) is a fluorograph of dissociated gp300 of HIV-2;

FIG. 3(b) and FIG. 3(c) are fluorographs of denatured gp300 of HIV-2;

FIG. 4 shows the result of electrophoresis of HIV-2 glycoproteins afterthe glycoproteins were digested with beta-N-acetylglucosaminidase H(endo H);

FIG. 5 is a fluorograph of a polyacrylamide gel after electrophoresis ofHIV-2 glycoproteins which were isotopically labeled with ¹⁴ C-mannose or³ H fucose;

FIG. 6 shows the result of electrophoresis of HIV-2 envelope proteinsobtained from cultures in which N-linked glycosylation was inhibited bythe antibiotic tunicamycin;

FIG. 7 is a fluorograph of a polyacrylamide gel after electrophoresis ofHIV-2 envelope glycoproteins obtained from cell cultures with andwithout oligosaccharide trimming inhibitors;

FIGS. 8A and 8B depict the results of electrophoresis of HIV-2glycoproteins obtained during pulse-chase experiments in HIV-2 infectedCEM cells in the absence (control) or presence of castanospermine (FIG.8A) or monensin (FIG. 8B);

FIG. 9 is a fluorograph of polyacrylamide gels after electrophoresis ofSIV envelope glycoproteins labeled with ³⁵ S-methionine, ³ H-fucose, or¹⁴ C-mannose; and

FIG. 10 is a schematic pathway postulated for in vivo processing ofHIV-2 envelope glycoprotein.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As a result of this invention, the processing of HIV-2 envelopeglycoproteins has now been characterized. Four glycoproteins referred toas gp300, gp140, gp125, and gp36 are synthesized in HIV-2 infectedcells. The gp125 and gp36 correspond to the external and transmembraneglycoproteins of HIV-2 virion, whereas gp300 and gp140 are onlydetectable in infected cells. The gp300 is a dimeric form of gp140,which is the immature precursor of HIV-2 envelope glycoprotein. Thisdimer is very stable since it resists ionic and non-ionic detergentshigh salt, 4M urea, and reducing agents. However, the dimer can bedissociated in acidic pH to yield gp140.

Dimerization occurs in the endoplasmic reticulum after the removal ofglucose residues by glucosidases I and II, and after the action of Golgimannosidases, the dimer becomes dissociated probably due to a shift inpH of the environment in trans Golgi. Finally, proteolytic cleavage ofthe mature precursor occurs outside the Golgi.

Transient dimer formation of the glycoprotein precursor seems to be anintrinsic property of the polypeptide moiety of HIV-2 envelope. This isa novelty in the mechanism of glycoprotein processing with N-linkedoligosaccharide chains. It is hypothesized that conformationalmodifications brought about by the formation of this dimer are necessaryfor transport of the glycoprotein precursor to the Golgi apparatus.

I. IDENTIFICATION OF THE HIV-2 ENVELOPE GLYCOPROTEINS

Recently, it has been reported that the envelope gene of HIV-2 (RODisolate) encodes a precursor glycoprotein that is then cleavedproteolytically to yield a 120Kd extracellular glycoprotein and a 36Kdtransmembrane glycoprotein (Clavel et al., 1986a and 1986b). To identifythe precursors of the HIV-2 glycoproteins, viral proteins in infectedcells as well as in virus particles were studied. For comparison, thesynthesis of HIV-1 proteins in cells infected with HIV-1 (BRU isolate)were also studied. The results are shown in FIG. 1 and were obtained asfollows.

A Comparison of high molecular weight proteins of HIV-1 and HIV-2 (FIG.1A)

CEM cells infected with HIV-1 or HIV-2 were labeled with ³⁵ S-methionine(200 μCi/ml; 4×10⁶ cells/ml) for 18 hours. Extracts from these infectedcells (CELL) and their corresponding culture medium (SN) were purifiedon specific immunoaffinity columns:

HIV-1 serum-Sepharose specific for HIV-1 proteins (Krust et al., 1988),and

HIV-2 serum-Sepharose specific for HIV-2 proteins.

(See "Experimental Procedures").

These purified proteins were analyzed by electrophoresis in a 7.5%polyacrylamide SDS-gel containing 6M urea. A fluorograph of the gel ispresented in FIG. 1. The sizes of the HIV-1 and HIV-2 proteins areindicated on the left and right of the lanes shown in FIG. 1.

Referring to FIG. 1A, the p68 and p55 are the reverse transcriptase andthe gag precursor, respectively. The gp160 and gp120 are theglycoprotein precursor of HIV-1 envelope and its cleaved product.

Three major high molecular weight glycoproteins of 300, 140, and 125Kdare detectable in HIV-2 infected cells (FIG. 1A). The proteins arespecific to HIV-2 because they are absent in non-infected cells andbecause they could be consistently identified by all HIV-2, but notHIV-1, seropositive sera in an immunoprecipitation assay (data notshown).

In side by side comparison, the electrophoretic mobility of these threeHIV-2 proteins is clearly different from that of the 160Kd HIV-1precursor glycoprotein (gp160) and one of its cleaved products, 120Kdexternal envelope glycoprotein (gp120; FIG. 1A). It should be noted thatthe resolution of the 140 and 125Kd proteins of HIV-2 from one anothercan be clearly observed in polyacrylamide-SDS gels containing a highconcentration of urea. In the absence of urea, these proteins migrate asa thick band. The 300 and 140Kd proteins are only detectable in infectedcells, hereas the 125Kd protein is detectable both in infected cells aswell as in the virus (FIG. 1A).

B. Identification of HIV-2 glycoproteins (FIG. 1B)

The glycosylation of the 300, 140, and 125Kd proteins was demonstratedby metabolic labeling with 3H-glucosamine (FIG. 1B). More particularly,HIV-2 CEM cells and T-lymphocytes were labeled with ³ H-glucosamine (200μCi/ml; 4×10⁶ cells/ml) for 18 hours. Extracts from infected cells(lanes C) and culture medium containing virus (lanes V) were purified onthe HIV-2 serum-Sepharose column. The labeled proteins were analyzed byelectrophoresis in a 7.5% gel. The lane on the far left depicts theresult of electrophoresis in a 12.5% acrylamide gel and shows thepresence of gp36. The gp36 is only slightly glycosylated and itsdetection required longer exposure times. Specifically, this part of theFigure had to be overexposed to see gp36; for this reason gp140/gp125are resolved as a thick band.

The presence of gp300 and gp140 is not restricted to infected CEM cells.They are also detectable in HIV-2 infected T4 lymphocytes as depicted inFIG. 1B. As in CEM cell cultures, gp300 and gp140 are detectable only ininfected cells, whereas gp125 is present both in cells and in HIV-2particles.

These results indicate that among the glycoproteins detectable in HIV-2infected cells, gp125 and gp36 correspond to the virion envelope,whereas gp300 and gp140 might be precursors of the envelopeglycoproteins.

C. Characterization of gp300 and gp140 (FIG. 2)

The proteins gp300, gp140, and gp125 were labeled with ³⁵ S-methionineand analyzed by two dimensional gel electrophoresis. The patterns ofresolution that were obtained indicated that gp300 and gp140 are closelyrelated.

More particularly, ³⁵ S-methionine labeled gp300, gp140, and gp125purified from HIV-2 infected CEM cells (CELL) and culture mediumcontaining virus (SN) prepared in the same manner as the experimentsreported in FIG. 1 were analyzed by two dimensional gel electrophoresis(See "Experimental Procedures"). The pH gradient obtained by isoelectricfocusing (first dimension) is shown in FIG. 2. In the second dimension,proteins were resolved on a 7.5% polyacrylamide-SDS gel containing 6Murea. Fluorographs of the gels are presented in FIG. 2.

Both proteins were resolved as an heterogeneous subspecies withidentical isoelectric points (pI) in the pH range of 6.8 to 7.8 (FIG.2). This similarity between gp140 and gp300 suggested that gp300 is adimeric form of gp140 (see below).

The gp125, which is present in both infected cells and in virusparticles, exhibited less heterogeneity and migrated with pI valuesbetween 6.2 to 6.5. In infected cells, there was a minor subspecies ofgp125 with a pI value of 7.2 to 7.3. This basic gp125 is notincorporated into the HIV-2 virion. Thus it might represent aglycoprotein that is not processed properly. The acidic nature of themature gp125 might be due to the addition of sialic acid on some of itscarbohydrate side chains during the processing of the envelopeglycoprotein.

D. Dissociation of the native (a) and the denatured (b and c) gp300(FIG. 3)

The gp300 is very stable since it resists ionic (1% SDS) and non-ionic(2% Triton X-100}detergents, urea (2-6M), high salt (1M NaCl), andreducing agents (1% β-mercaptoethanol). However, it was possible todemonstrate that gp300 could be dissociated into gp140 in acidic pH. Inthese experiments, immunoaffinity column bound proteins were incubatedin acetate buffer at pH values varying between 4 to 7. These sampleswere then analyzed by polyacrylamide gel electrophoresis. Fluorographsof the gels are shown in (a), (b), and (c) in FIG. 3. In section (c),the band of gp300 and the dissociated gp140 were quantified bydensitometric scanning of the fluorograph. More particularly, the gelswere prepared as follows.

(a) ³⁵ S-methionine labeled extracts from HIV-2 infected CEM cells werepurified on the HIV-2 serum-Sepharose column. This sample was thendivided into two equal aliquots: one was incubated in the bindingbuffer, FIG. 3(a), lane 1, whereas the other one was incubated in buffercontaining 30 mM sodium acetate pH 4.0, 0.2mM PMSF, 100 units/mlaprotinin and 5 mM β-mercaptoethanol, FIG. 3(a), lane 2. After 1 hour at37° C., the acidic medium was neutralized and both samples were analyzedby electrophoresis.

(b) Purified and lyophilized ³⁵ S-methionine labeled gp300 was suspendedin 100 μl of the sodium acetate buffer pH 4.0 as above FIG. 3(b), lane2. Incubations were carried out for 30 minutes at 37° C. before additionof 2-fold electrophoresis sample buffer containing 2M urea. In lane 1 ofFIG. 3(b), the lyophilized gp300 was directly suspended in theelectrophoresis sample buffer.

(c) The purified and lyophilized ³⁵ S-methionine labeled gp300 wassuspended in solution containing 30mM Tris-HCl, 0.2mM PMSF and 100units/ml aprotinin and buffered with HCl at pH 7.5, 7.0, 6.5 and 6.0 (asindicated). After 60 minutes at 37° C., two fold electrophoresis samplebuffer was added and the samples were analyzed by electrophoresis.

FIG. 3(a) shows that the band of gp300 shifted to the position of gp140when the sample was incubated at pH 4. Further experiments were carriedout using purified preparations of gp300 obtained by preparative gelelectrophoresis. Such denatured samples of gp300 were dissociatedcompletely in acetate buffer at pH 6.0, FIG. 3(b). The efficiency ofdissociation of the purified gp300 was probably due to a decrease in thepH along with the presence of residual SDS in the lyophilized sample,since column-bound native gp300 was not dissociable in the same bufferat pH values higher than pH 5 (data not shown).

In Tris-HCl buffer, the dissociation was less efficient. At pH 7.5 therewas only a slight dissociation of gp300 to gp140, but it increased withdecreasing pH values. In Tris buffer at pH 6.0, the dissociation wasabout 80%, (FIG. 3(c). During dissociation of the pure gp300 in eitheracetate or Tris-HCl buffer no proteins other than gp140 were detectable(experiments carried out in 15% polyacrylamide gels; data not shown).

These results indicate that gp300 is a dimeric form of gp140, theprecursor of HIV-2 envelope glycoprotein. Thus, it seems most likelythat during the processing of the envelope glycoprotein, two moleculesof gp140 become fused by a pH-dependent mechanism.

E. Characterization of the Oligosaccharide Side Chains of HIV-2Glycoproteins (FIGS. 4 and 5)

Digestion with endo β-N-acetylglucosaminidase H (endo H) demonstratedthe presence of N-linked oligosaccharides of the high mannose type onHIV-2 glycoproteins. The gp300, and the gp140+gp125, and the gp125 werepurified by immunoaffinity chromatography and preparativeelectrophoresis. (See "Experimental Procedures"). The lyophilizedsamples were suspended in endo H digestion buffer which does not promotethe dissociation of gp300 to gp140. The procedure was carried out asfollows.

Purified and lyophilized gp300, gp140/gp125 and gp125 ("ExperimentalProcedures") were suspended in buffer containing 150 mM sodium citratepH 5.5, 0.1% SDS (w/v), 0.5 mM PMSF before heating for 2 minutes at 90°C. Aliquots of these samples were then incubated (2 hours, 30° C.)without (lanes 1, FIG. 4), or with 0.4 milli-units of endo-H (lanes 2,FIG. 4), 2 milli-units of endo-H (lanes 3, FIG. 4), and 10 (lanes 4,FIG. 4) milli-units of endo-H. All the reactions were stopped by theaddition of two fold electrophoresis sample buffer. Electrophoresis wasas previously described in relation to FIG. 1. Fluorographs of thedifferent gels are shown in FIG. 4. The arrows p90 and p80 on the rightindicate the position of the digested product. Conditions for endo-Hdigestion were as described (Tarentino et al., 1974).

Upon endo H digestion, the electrophoretic mobility of gp300 was reducedto a protein of 200-250Kd A small fraction of gp300 that had becomedissociated into gp140, was digested to give rise to a 80Kd protein(FIG. 4, section gp300, lane 4).

The gp140 +gp125 sample was digested by endo H into 90 and 80Kd proteinswhereas gp125 was converted into a 90Kd protein (FIG. 4, sectionsgp140/125 and gp125). These results indicate that endo H digestion ofgp140 and gp125 give products of molecular weight 80 and 90Kd,respectively. The resistance to endo H digestion of gp125 relative togp140 is probably due to the conversion of some high mannose typeoligosaccharide side chains into complex oligosaccharides duringprocessing of the envelope glycoprotein (Kornfeld and Kornfeld, 1985).

Metabolic labeling of cells was carried out with ¹⁴ C-mannose and ³H-fucose. More particularly, HIV-2 infected CEM cells were labeled (18hours) with ¹⁴ C-mannose 25 μCi/ml; 4×10⁶ cells/ml) or ³ H-fucose (200μCi/ml; 4×10⁶ cells/ml). Extracts from infected cells (lanes C, FIG. 5)and culture medium containing virus (lanes V, FIG. 5) were purified onHIV-2 serum-Sepharose. Labeled glycoproteins were then analyzed bypolyacrylamide gel electrophoresis. A fluorograph is shown in FIG. 5.

Referring to FIG. 5, it will be apparent that metabolic labelingresulted in the incorporation of mannose into gp300, gp140, and gp125whereas only gp300 and gp125 were able to incorporate fucose. Fucoseresidues are normally transferred on oligosaccharide chains late in theglycosylation cycle, after the action of trimming enzymes of theendoplasmic reticulum and Golgi apparatus (Kornfeld and Kornfeld, 1985;Fuhrmann et al., 1985). The fact that gp140 does not contain fucoseresidues was consistent with it being the precursor of gp300 and gp125.

F. The Effect of Glycosylation Inhibitor Tunicamycin on the Processingof HIV-2 Glycoproteins (FIG. 6)

All glycoproteins carrying N-linked glycans derive their oligosaccharidemoiety from the lipid-linked oligosaccharide, Glc₃ Man_(q) -GlcNAc₂-pp-Dolichol, through a reaction carried out by protein-oligosaccharidyltransferase, which catalyzes the en bloc transfer of oligosaccharidechains to asparagine residues (for references, see Kornfeld andKornfeld, 1985). Tunicamycin blocks such N-linked glycosylation since itinhibits the production of N-acetylglucosamine pyrophosphoryldolichol,the first step in the assembly of lipid-linked oligosaccharides (Li etal, 1978; Heifetz et al., 1979).

In the presence of 2 μg/ml tunicamycin, the overall N-linkedglycosylation of HIV-2 envelope glycoproteins was completely blocked ininfected CEM cells. This was demonstrated by the lack of ³ H-glucosamineincorporation in viral glycoproteins, gp300, gp140, and gp125.Inhibition of N-linked glycosylation by tunicamycin was carried out asfollows.

HIV-2 infected cells in the absence (lanes C, FIG. 6) or presence (TM,FIG. 6) of tunicamycin (2 μg/ml) were labeled with ³⁵ S-methionine(panel "³⁵ S-met"; 200 μCi/ml; 4×10⁶ cells/ml) or with ³ H-glucosamine(panel "³ H-GLcNAc"; 200 μCi/ml; 4×10⁶ cells/ml) for 16 hours. Cellstreated with tunicamycin were first incubated (37° C.) with theantibiotic (2 μg/ml) for 2 hours before labeling with ³⁵ S-methionine or³ H-glucosamine. Extracts from infected cells (CELL) and from theculture medium containing virus (SN) were purified by HIV-2serum-Sepharose and analyzed by polyacrylamide 7.5% gel electrophoresis.Fluorographs of the gels are presented in FIG. 6. The position of theunglycosylated envelope precursor (p90/80) and the unglycosylated dimer(200Kd) are indicated by the small arrows on the right. These 90/80Kdand 200Kd proteins do not incorporate ³ H-glucosamine (panel ³ H-GlcNAc,cell lane TM).

Under these experimental conditions, protein synthesis was not affectedin infected cells treated with tunicamycin (data not shown). Suchcultures isotopically labeled with ³⁵ S-methionine accumulated two majorproteins of apparent sizes, 200 and 80-90Kd, which migrated as widebands (FIG. 6). The molecular weight of these proteins coincides wellwith endo H digestion products of gp300, gp140 and gp125 (FIG. 4), thussuggesting that the 200 and 80-90Kd proteins correspond tounglycosylated forms of HIV-2 envelope glycoproteins. The molecularweight of the 80-90Kd protein corresponds to the expected molecularweight of unglycosylated HIV-2 envelope precursor estimated from itsnucleic acid sequence (Guyader et al., 1987). The 200Kd protein isprobably the dimeric form of the unglycosylated envelope precursor.These results confirm that HIV-2 envelope proteins have N-linkedpolysaccharide chains.

Besides inhibition of glosylation, tunicamycin treatment inhibits theprocessing and export of the envelope glycoprotein since the 80-90Kdprotein was not found in the extracellular medium (FIG. 6, lanes SN).Oligosaccharide chains of HIV-2 envelope proteins, therefore, areprobably involved in the cellular transport through the Golgi apparatus.The absence of unglycosylated forms of the envelope protein in theextracellular medium of tunicamycin treated cells might also be due toits rapid degradation. Several reports have suggested that theunglycosylated form of a protein is more sensitive to proteases than itsglycosylated form (Olden et al., 1978; Schwartz et al., 1976).Accordingly, the small molecular weight proteins in ³⁵ S-methioninelabeled cells cultured with tunicamycin might represent partiallydegraded products of the unglycosylated envelope protein (FIG. 6).

G. Effect of Oligosaccharide Trimming Inhibitors on the Synthesis ofHIV-2 Glycoproteins (FIG. 7)

Asparagine-linked oligosaccharides (Glc₃ Man_(q) GlcNac₂) ofglycoproteins undergo extensive modifications or processing followingtheir attachment to nascent proteins (reviewed by Kornfeld and Kornfeld,1985). The trimming reactions occur in the lumen of the roughendoplasmic reticulum (RER) and in the Golgi apparatus by specificglucosidases and mannosidases.

Processing of oligosaccharide chains of glycoproteins can be manipulatedwith the aid of specific inhibitors of the trimming glucosidases andmannosidases (reviewed by Schwarz and Datema, 1984; Fuhrmann et al.,1985). In these experiments, different trimming inhibitors were used toinvestigate the localization of HIV-2 glycoprotein precursors and alsoto study the role of glycosylation in the processing of the envelopeprecursor. The inhibitors used were:

castanospermine, a plant alkaloid that inhibits glucosidase I (Saul etal., 1983);

deoxynojirimycin (dNM), a glucose analogue that inhibits trimmingglucosidase I and II (Lemansky et al., 1984);

1-deoxymannojirimycin (dMM), a mannose analogue that inhibitsmannosidase catalyzed reactions (Fuhrmann et al., 1984);

bromoconduritol (6-bromo-3,4,5-trihydroxycyclohex-1-ene) that inhibitsglucosidase II (Datema et al., 1982); and

swainsonine, an indolizidine alkaloid that inhibits Golgi mannosidase II(Tulsiani et al., 1982).

Specifically, HIV-2 infected CEM cells were labeled (16 hours, 37° C.)with ³⁵ S-methionine (200 μCi/ml; 4×10⁶ cells/ml) in the absence (lanesT, FIG. 7) or presence of the oligosaccharide trimming inhibitor

1 mM bromoconduritol (lanes Bro, FIG. 7);

1 mM castanospermine (lanes Cast, FIG. 7);

10 μg/ml swainsonine (lanes Sw, FIG. 7);

3 mM deoxynojirimycin (lanes dNM, FIG. 7); and

1 mM deoxymannojirimycin (lanes dMM, FIG. 7).

Extracts from infected cells (panel CELL) and from culture mediumcontaining virus particles (panel SN) were purified on HIV-2serum-Sepharose to identify viral glycoproteins gp125, gp140, and gp300in infected cells and gp125 in culture medium. All samples were analyzedby polyacrylamide (7.5%) gel electrophoresis.

In order to show that inhibition of gp125 production by cells treatedwith different inhibitors is specific to the viral glycoprotein, culturemedia were assayed for viral core protein p26 by an immunoprecipitationassay using an HIV-2 seropositive serum (Clavel et al., 1986a, 1987).The p26 was analyzed by polyacrylamide (12.5%) gel electrophoresis. FIG.7 represents a fluorograph showing only one part of each gel.

As expected, control infected cells contained gp300, gp140, and gp125whereas only gp125 was observed in the extracellular medium (FIG. 7,sections cell and SN, lanes T). In cells treated with castanospermine ordNM, there was a normal level of gp300, no gp125 and a small amount of a150Kd protein that probably corresponds to the glucosylated form ofgp140. In such cells, therefore, the processing of the envelopeglycoprotein was blocked since no gp125 was detectable in theextracellular medium in spite of the production of p26, the core proteinof HIV-2 (FIG. 7, lanes Cast and dNM). These results indicate thatremoval of the terminal glucose residues from the oligosaccharide chainsof the envelope glycoprotein precursor is necessary for its processingand cleavage by the cellular protease.

Bromoconduritol, which acts on glucosidase II, also inhibited by 70-90%the normal production of gp125, but the levels of gp140 and gp300remained normal (FIG. 7, lanes Bro). In contrast to castanospermine anddNM (which inhibit removal of terminal glucose residue), bromoconduritoltreatment (which inhibits removal of two inner glucose residues) did notblock completely the processing of HIV-2 envelope glycoprotein. In fact,low amounts of gp125 were detectable intracellularly andextracellularly. This latter result suggests that a low level of mannosetrimming can occur without removal of the two inner glucose residues.Such a phenomenon has been observed previously for the processing ofother viral glycoproteins during bromoconduritol treatment (Datema etal., 1982).

Mannosidase inhibitors, swainsonine and dMM, did not cause an apparentmodification in the level of intracellular gp300, gp140, and gp125, butthe level of extracellular gp125 was 50% less than that from thecorresponding control cells (FIG. 7, lanes Sw and dMM). Thus, althoughthe oligosaccharide chain was only deglucosylated, the glycoproteinprecursor was proteolytically cleaved to yield a protein similar togp125 but with a higher content of mannose, which probably affected thecellular transport of gp125. The molecular weight of the extracellularglycoprotein produced in the presence of dMM was slightly higher thanthat produced in the absence of the inhibitor. This is probably due tothe higher content of mannose residues in the extracellular proteinsynthesized by dMM-treated cells (FIG. 7, section SN).

It should be emphasized that the effects of trimming enzyme inhibitorson the processing of HIV-2 envelope glycoprotein were specific Since thesynthesis (data not shown) and the production of HIV-2 p26 was notaffected at all (FIG. 7, section SN).

H. Effect of Castanospermine and Monensin on the Processing of HIV-2Glycoproteins (FIG. 8)

To study the intracellular processing of HIV-2 glycoproteins,pulse-chase experiments were performed. The results are shown in FIG. 8.More particularly, the experiments were carried out as follows:

(a) Pulse-chase experiments were performed in HIV-2 infected CEM cellsin the absence (Control) or presence of 1 mM castanospermine (Cast.).Control: infected cells were incubated 1 hour at 37° C. inmethionine-free medium before 15 minutes pulse labeling with ³⁵S-methionine (200 μCi/ml; 4×10⁵ cells/ml; lane 1, FIG. 8a). Theradioactive label was then chased in culture medium containing 5mM coldmethionine for 0.5, 1.5, and 3 hours (in lanes 2, 3 and 4, respectively,FIG. 8a). Cast.: HIV-2 infected CEM cells were incubated (1 hour, 37°C.) in methionine-free medium containing castanospermine before 30minutes pulse labeling with ³⁵ S-methionine (lane 1, FIG. 8a). Thesecells were then chased as above, but in the presence of castanosperminefor 0.5, 1.5, and 3 hours (lanes 2, 3 and 4, respectively, FIG. 8a).

The gp140 was the first protein detectable 15 minutes after pulselabeling. During the chase, gp300 became detectable at 0.5 hours,whereas gp125 became detectable at 1.5-3 hours. The fact that gp300 wasobserved after synthesis of gp140 and the fact that gp125 was detectableonly after formation of gp300 (FIG. 8A, lanes 1-4), suggest thatdimerization is an intermediate step necessary for the oligosaccharideprocessing towards the mature glycoprotein, gp125. This suggestion wasconfirmed by the use of castanospermine, which inhibits the trimming ofthe external glucose residue of polysaccharide chains.

After 30 minutes of pulse labeling in the presence of castanospermine, a150Kd protein was detectable along with gp300 (FIG. 8, Cast., lane 1).The 150Kd protein should correspond to gp140; the slight increase in themolecular weight of the first precursor is ascribed to the presence ofglucose residues in its oligosaccharide chains. Thus, gp140 synthesizedin HIV-2 infected cells represents the precursor glycoprotein withoutits glucose residues. Accordingly, the 150Kd protein (gp150) representsthe first immature glycoprotein of HIV-2 envelope. The removal ofglucose residues in control cells has been reported to be a rapidprocess occurring during or briefly after cotranslational translocationof precursor glycoproteins into endoplasmic reticulum (Lemansly et al.,1984). After 30 minutes of pulse and 3 hours of chase in the presence ofcastanospermine, the level of gp150 was gradually reduced while gp300accumulated (FIG. 8, Cast, lanes 1-4). Under these conditions, theprecursor was not cleaved to yield gp125.

Further characterization of HIV-2 envelope glycoprotein was studied inpulse-chase experiments using monensin, a cationic ionophore thatinhibits the transport of proteins from Golgi to the plasma membrane orin some cases it might even block the transport of proteins at the levelof the medial Golgi cisternae (Tartakoff and Vassali., 1977; Johnson andSchlesinger, 1980; Strous and Lodish, 1980; Griffiths et al., 1983).HIV-2 infected cells in the absence or presence of monensin were pulsedlabeled as follows:

(b) Pulse chase experiments in HIV-2 infected cells were carried out inthe absence (Control) or presence of 1 μM monensin. Infected cells withor without monensin were incubated (1 hour, 37° C.) in methionine-freemedium before 30 minutes pulse labeling with ³⁵ S-methionine (lanes 1,FIG. 8b). Labeled cells were then chased in culture medium containing 5mM cold methionine for 0.5, 1.5, and 3 hours (lanes 2, 3 and 4,respectively, FIG. 8b). Extracts were purified on HIV-2 serum-Sepharose,and labeled proteins were analyzed by polyacrylamide (7.5%) gelelectrophoresis. Fluorographs are shown in FIG. 8b. (The p55 shows thegag precursor in section A, lanes 1.)

In the presence of monensin, HIV-2 infected cells synthesized normallevels of gp140 and its dimeric form. However, no gp125 was detectablein monensin treated cells. After 1.5-3 hours of chase, monensin treatedcells accumulated a 135Kd protein (gp135) that is probably thedissociated product of the dimer precursor. The slightly smallermolecular weight of gp135 might be accounted for by the removal of somemannose residues by the action of RER and Golgi mannosidases. In view ofthese results, it is tempting to speculate that after deglucosylation,gp300 becomes trimmed by mannosidases of RER and Golgi before itsdissociation into the mature precursor gp135 of HIV-2 envelope. Thisgp135 could then be transported to plasma membrane and also be cleavedby cellular protease. Inhibition of protein transport by monensin blocksthe mature glycoprotein gp135 in trans Golgi. No mature envelopeglycoproteins are detectable in monensin treated cells intracellularlyor extracellularly, although p26 is synthesized and excreted (data notshown).

I. Dimerization of the Glycoprotein Precursor Occurs also in SIVmacInfected Cells (FIG. 9)

The nucleotide sequence of HIV-2 envelope shows a considerable homology(75% amino acid identity) to that of SIV (Guyader et al., 1987;Chakrabarti et al., 1987; Franchini et al., 1987). For this reason, itwas important to investigate whether dimerization of envelopeglycoproteins is detectable in SIV infected cells. SIV proteins werepurified by the immunoaffinity column containing antibodies specific forHIV-2 proteins, since the gag, pol, and env proteins of HIV-2 and SIVare antigenically cross-reactive.

More particularly, SIV-infected HUT-78 cells were labeled (16 hours, 37°C.) with ³⁵ S-methionine (200 μCi/μl; 4×10⁶ cells/ml), ³ H-fucose (200μCi/μl; 4×10⁶ cells/ml) and ¹⁴ C-mannose (25 μCi//μl; 4×10⁶ cells/ml).Extracts from infected cells (lanes C, FIG. 9) and from the culturemedium containing SIV (lanes V, FIG. 9) were purified on HIV-2serum-Sepharose. Because of cross-reactivity between HIV-2 and SIVproteins, the HIV-2 positive serum could be used to immunoprecipitateSIV proteins. All samples were analyzed by polyacrylamide (7.5%) gelelectrophoresis. (See "Experimental procedures".) A fluorograph of thedifferent gels is shown in FIG. 9.

FIG. 9 shows that SIV infected cells synthesize three high molecularweight proteins analogous to those synthesized in HIV-2 infected cells:gp300, gp140, and gp130. The electrophoretic mobility of gp300SIV andgp140SIV correspond to that of HIV-2 glycoproteins gp300 and gp140 (datanot shown). The gp130SIV has a slightly higher mobility than gp125 ofHIV-2. The p55 labeled with ³⁵ S-methionine is probably the gagprecursor of SIV.

Evidence that these proteins present in SIV infected cells areglycoproteins was provided by the isotopic labeling with ¹⁴ C-mannoseand ³ H-fucose. All the three proteins incorporated mannose, but onlygp300_(SIV) and gp130_(SIV) incorporated fucose (FIG. 9). Thegp300_(SIV) and gp140_(SIV) are intracellular proteins, whereasgp130_(SIV) is the extracellular glycoprotein. The fact that gp300_(SIV)and gp130_(SIV) can incorporate fucose suggest that they are processedproducts of gp140_(SIV).

These results indicate that doublet formation of the envelopeglycoprotein precursor is a specific property of HIV-2 and SIV envelopegene expression. It should be emphasized that HIV-1 envelopeglycoprotein does not undergo dimerization during its processing. HIV-1infected cells in the presence of castanospermine or dNM do notaccumulate envelope dimers (data not shown) as it is the case for HIV-2or SIV.

This invention thus describes for the first time the processing of HIV-2envelope glycoproteins and details a novel mechanism of glycoproteinprocessing with N-linked oligosaccharide chains. The envelopeglycoproteins of HIV-2, i.e. the extracellular gp125 and transmembranegp36, arise from a common precursor glycoprotein (Guyader et al., 1987).The unusual feature of this glycoprotein precursor is that it requiresthe formation of a homologous dimer in order to become transported andprocessed through the Golgi apparatus. The mechanism of dimerization ofenvelope glycoprotein is not entirely clear. The fact that the purifieddimer can be dissociated at an acidic pH (pH 6.0) suggests thatdimerization might be pH dependent. Oligosaccharide chains on theprecursor glycoprotein are not essential for dimer formation. Evidencefor this has been obtained by two different experiments: (1) Digestionwith endo H results in a shift in the electrophoretic mobility of thedimer without dissociating it; and (2). In the presence of tunicamycin,HIV-2 infected cells synthesize an unglycosylated envelope precursor(80-90Kd) that can form a dimer (200Kd). These results emphasize thatthe dimer formation is an intrinsic property of the polypeptide moietyof the envelope precursor.

Pulse-chase experiments in the absence or presence of castanospermine(FIG. 8) suggest that dimerization of the glycoprotein precursornormally occurs immediately after removal of glucose residues. Sinceglucosidases are associated with membranes of endoplasmic reticulum,then it is most likely that dimerization occurs in the RER. In thepresence of castanospermine, the dimer becomes accumulated in RER and itis not processed. However, once the glucose residues are removed, theninhibition of the RER mannosidase does not prevent the processing of theglycoprotein-dimer through the Golgi apparatus (FIG. 7). Accordingly,the glucose residues in the oligosaccharide chains of the dimerprecursor prevent its exit from the RER. In accord with this, it hasbeen postulated that glucose trimming is necessary for efficienttransport from the RER to the Golgi, possibly because the deglucosylatedoligosaccharide forms part of a recognition site for a transportreceptor (Lodish and Kong, 1984; Lemansky et al., 1984). It might alsobe possible that glucose removal is crucial for the precursor dimer toachieve a correct functional configuration (Schlesinger et al., 1984)that favors the action of trimming mannosidases.

In view of these results, a schematic pathway for the processing ofHIV-2 envelope glycoproteins is proposed in FIG. 10. With reference toFIG. 10, the expected size of the polypeptide moiety of the precursorenvelope glycoprotein is about 80Kd (FIGS. 4 and 6). The oligosaccharidechain is transferred from dolichol-P-P to the newly synthesized envelopeprecursor (80Kd) probably at acceptor amino-acid asparagine residues(Kornfeld and Kornfeld, 1985). As depicted in FIG. 10, tunicamycininhibits assembly of dolichol-P-P glycan, and for this reason the 80Kdprotein does not become glycosylated. Addition of oligosaccharide chainsto the 80Kd protein yields the first envelope glycoprotein precursor,gp150. This precursor might or might not exist as such in infectedcells, since addition of polysaccharide chains and glucose trimmingprobably occurs during translation of the precursor. Whatever is thecase, gp150 becomes rapidly deglucosylated to give gp140. At this stage,a difference in environment, perhaps of pH, would trigger dimerformation by the fusion of two gp140 molecules. The resulting gp300 canthen be trimmed by the RER mannosidase and transported to the Golgiapparatus. In the presence of castanospermine or dNM, gp150 becomesdimerized and is accumulated in the RER. This dimer is not processedbecause it is glucosylated. However, as long as the dimer is found inthe deglucosylated form, it can be transported to the Golgi; inhibitionof RER mannosidase by dMM does not block processing of the dimerprecursor. In the Golgi, gp300 traverses the different compartmentsprobably by vesicular transport (Griffiths and Simons, 1986) duringwhich the oligosaccharide chain is further trimmed by Golgi mannosidasesbefore addition of other sugars such as fucose and sialic acid. Evidencefor fucose incorporation has been obtained by isotopic labeling of gp300with ³ H-fucose. Evidence for sialic acid incorporation was obtainedindirectly by digesting gp300 with neuraminidase, an enzyme thathydrolyzes terminal N-acetylneuraminic acid in various glycoproteins(Peyrieras et al., 1983). The gp300 of HIV-2 is susceptible to digestionwith neuraminidase as evidenced by a significant decrease in theelectrophoretic mobility of the dimer (data not shown). The results areconsistent with the precursor keeping its dimeric form all through itsprocessing in the Golgi cis, medial, and trans cisternae before itstransport to the trans-Golgi network (TGN; Griffiths and Simons, 1986)where it dissociates due to a drop in the pH of this compartment. Thedissociated dimer yields glycoproteins (gp135) of slightly smallermolecular weight than the first detectable glycoprotein precursor(gp150-140). The gp135 could then be transported to plasma membrane andalso be cleaved by the cellular protease to yield the matureglycoproteins of HIV-2 envelope, gp125, and gp36. Monensin most probablyinhibits transport from the Golgi to the plasma membrane; for thisreason gp135 accumulates in the Golgi.

It is well accepted that the Golgi apparatus is implicated in themechanism of sorting secretory and plasma membrane proteins, which seemsto take place in the last Golgi compartment referred to as TGN(Griffiths and Simons, 1986). This compartment on the trans side of theGolgi stack, previously has been referred to as Golgi endoplasmicreticulum lysosomes (GERL) and recently as post-Golgi vacuoles or thetrans-most cisternae of the Golgi stack (Novikoff, 1976; Saraste andKuismanen, 1984; Orci et al., 1987). Interestingly, the pH of the TGNhas been considered to be mildly acidic, i.e., about pH 6 (Anderson andPathak, 1985; Griffiths and Simons, 1986). The acidic pH in the TGNcould then account for the dissociation of the processed dimer.

The results discussed here illustrate that the processing of theenvelope glycoproteins of HIV-2 is a multistep process involving thesynthesis of an immature precursor gp150-140, the intermediary dimerprecursor gp300 and finally the mature precursor gp135. Despite theirevolutionary relationship, HIV-1 and HIV-2 have found differentmechanisms for the processing of their envelope glycoproteins. Whetheror not these differences are involved in their pathogenesis is underinvestigation.

Following is a more detailed description of the experimental proceduresused in this invention.

II. EXPERIMENTAL PROCEDURES A. Materials

L-(³⁵ S)Methionine (specific activity 1000 Ci/mmol, L-(6-³ H) Fucose(specific activity: 45-70 Ci/mmol), D-(6-³ H)Glucosamine (specificactivity: 20-40 Ci/mmol and D-(U-¹⁴ C)Mannose (specific activity:200-300 mCi/mmol) were purchased from Amersham (Amersham, UK).Bromoconduritol, castanospermine, 1-deoxymannojirimycin (dMM),1-deoxynojirimycyn (dNM), swainsonine and tunicamycin were obtained fromBoehringer-Mannheim (Mannheim, West Germany). EndoB-N-acetylglucosaminidase H was from Calbiochem (San Diego, USA).Ampholines were purchased from Pharmacia (Uppsala, Sweden).

B. Virus and Cells

HIV-1_(BRU) isolate of the human immunodeficiency virus type 1(Montagnier et al., 1984), HIV-2_(ROD) isolate of the humanimmunodeficiency virus type 2 (Clavel et al., 1986a), and Simianimmunodeficiency virus, SIVmac₁₄₂ (Daniel et al., 1985), were used inthis study.

The different cell lines and human lymphocytes were cultured insuspension medium RPMI-1640 (GIBCO-BRL, Cergy-Pontoise, France)containing 10% (v/v) fetal calf serum; 2 μg/ml polybrene (Sigma) wasadded for HIV infected cell cultures. CEM clone 13 cells are derivedfrom the human lymphoid cell line CEM (ATCC-CCL119) and express the T4antigen to a high level. Five days after infection with HIV-1_(BRU) orHIV-2_(ROD) isolates, about 80-90% of the cells produce viral particlesand can be identified by a cytopathic effect corresponding tovacuolization of cells and appearance of small syncitia.

The HUT-78 cell line is another human T4 positive lymphoid cell line(Gadzudar et al., 1980) that is highly permissive for the replication ofSIVmac₁₄₂ (Daniel et al., 1985). Peripheral blood lymphocytes fromhealthy blood donors were stimulated for three days with 0.2% (w/v)phytohemagglutinin fraction P (Difco, Detroit, USA) in RPMI-1640 mediumsupplemented with 10% fetal calf serum. Cells were then cultured inRPMI-1640 medium containing 10% (v/v) T cell growth factor (TCGF,Biotest). After infection with HIV-2, lymphocytes were cultured inpresence of 10% (v/v) TCGF and 2 μg/ml Polybrene.

C. Metabolic Labeling of Cells

For metabolic labeling of proteins, infected cells were incubated for 16hours at 37° C. in MEM culture medium without L-methionine and serum butsupplemented with 200 μCi/ml ³⁵ S-methionine. For metabolic labeling ofglycoproteins, infected cells were incubated for 16 hours at 37° C. inMEM culture medium lacking serum and glucose but supplemented with 200μCi/ml ³ H-fucose or 200 μCi/ml ³ H-glucosamine or 25 μCi/ml ¹⁴C-mannose.

D. Cell and Viral Extracts

Cell pellets corresponding to 10⁷ cells were resuspended in 100 μl ofbuffer: 10 mM Tris-HCl pH 7.6, 150 mM NaCl, 1 mM EDTA, 0.2 mM PMSF, 100units/ml aprotinin (Iniprol, Choay) before addition of 100 μl of thesame buffer containing 2% (v/v) Triton X-100. Cell extracts werecentrifuged at 12,000 g for 10 minutes, and the supernatant was storedat -80° C. until used. For viral extract preparations, 100 μl of 10×lysis buffer (100 mM Tris-HCl pH 7.6, 1.5 M NaCl, 10 mM EDTA, 10% (v/v)Triton X-100, 100 units/ml aprotinin) was added per ml of clarifiedsupernatant from infected CEM cells and processed as above.

E. Preparation of an Immunoadsorbant with Antibodies from an HIV-2Seropositive Patient Sera

Immunoglobulins from the serum of an HIV-2 seropositive patient wereprecipitated with 50% (NH dissolved in 20 mM sodium phosphate (pH 8.0)and further purified on a DEAE cellulose column (DE 52, Whatman) byelution with 20 mM sodium phosphate (pH 8.0). Immunoglobulins purifiedin this manner were judged to be 90% pure. The antibodies weresubsequently coupled to CNBr-activated Sepharose CL 4B according to atechnique described by Berg (1977). Two milligrams of IgG were coupledper ml of Sepharose CL 4B. This immunoadsorbant is referred to as HIV-2serum-Sepharose.

F. Binding of the HIV-2 Proteins on the Immunoaffinity Column

Cell extracts from HIV-2 producing CEM cells were first diluted in twovolumes of binding buffer (20 mM Tris-HCl pH 7.6, 50 mM KCl, 150 mMNaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 20% (v/v) glycerol, 7mM-mercaptoethanol, 0.2 mM PMSF, 100 units/ml aprotinin) beforeincubation with one volume of HIV-2 serum-Sepharose. Supernatants fromHIV-2 producing cells were processed as cell extracts except that onlyone tenth of binding buffer concentrate 10× was added per volume ofsupernatant. The binding was carried out overnight, then the column waswashed batchwise in binding buffer. Proteins bound to the column wereeluted by boiling in electrophoresis sample buffer (125 mM Tris-HCl pH6.8, 1% (w/v) SDS, 2M urea, 20% glycerol, 1% β-mercaptoethanol. Elutedproteins were resolved by electrophoresis on 7.5% polyacrylamide-SDSgels containing 6M urea and 0.1% bisacrylamide instead of 0.2% (w/v).

G. Preparative Electrophoresis

HIV-2 glycoproteins eluted from the affinity column were resolved bypolyacrylamide gel electrophoresis as previously described, and theregions of the gel containing the viral glycoproteins were cut out byreference to the position of prestained molecular weight protein markers(BRL).

Glycoproteins were eluted by incubation for 16 hours at 4° C. in elutionbuffer (0.1M NaHCO₃, 0.5 mM EDTA, 0.05% (w/v) SDS, 0.2 mM PMSF). Theglycoprotein fractions thus obtained were lyophilized and keptrefrigerated until used.

H. Two Dimensional Electrophoresis

Two dimensional gel electrophoresis was performed as described byO'Farrel (1975) with the following modification: L-(³⁵ S)-methioninelabeled proteins bound on the HIV-2 serum-Sepharose column were elutedby boiling in electrophoresis sample buffer as previously describedbefore dilution in a volume of buffer containing 9.5M urea, 8%(v/v)-mercaptoethanol, 1.6% (w/v) ampholines pH ranges 6.5-9, 0.4% (w/v)ampholines pH ranges 3-10 and 100 units/ml aprotinin.

It will be understood that the present invention is intended toencompass the previously described proteins and glycoproteins inpurified form, whether or not fully glycosylated, and whether obtainedusing the techniques described herein or other methods. In a preferredembodiment of this invention, the polypeptides are substantially free ofhuman tissue and human tissue components, nucleic acids, extraneousproteins and lipids, and adventitious microorganisms, such as bacteriaand viruses. It will also be understood that the invention encompassesequivalent proteins and glycoproteins having substantially the samebiological and immunogenic properties. Thus, this invention is intendedto cover serotypic variants of the proteins and glycoproteins of theinvention.

The proteins and glycoproteins of this invention can be obtained byculturing HIV-2 in susceptible mammalian cells of lymphocytic lineage,such as T-lymphocytes or pre-T-lymphocytes of human origin or non-humanprimate origin (e.g. chimpanzee, African green monkey, or macaques.) Anumber of different lymphocytes expressing the CD4 phenotypic marker canbe employed. Examples of suitable target cells for HIV-2 infection aremononuclear cells prepared from peripheral blood, bone marrow, and othertissues from patients and donors. Alternatively, established cell linescan be employed. For example, HIV-2 can be propagated on blood-donorlymphocyte cultures, followed by propagation on continuous cell strainsof leukemic origin, such as HUT 78. HUT 78 is a well characterizedmature human T cell line, which has been deposited at CollectionNationale Des Cultures De Micro-organismes (CNCM) at the PasteurInstitut in Paris, France on Feb. 6, 1986, under culture collectiondeposit accession number CNCM I-519. Another suitable target for HIV-2infection and production of the proteins and glycoproteins of theinvention is the T-cell line derived from an adult with lymphoidleukemia and termed HT. HT cells continuously produce virus afterparental cells are repeatedly exposed to concentrated cell culturefluids harvested from short-term culture T-cells grown in TCGF thatoriginated from patients with LAS or AIDS. In addition, there areseveral other T or pre-T human cell lines, such as CEM and MOLT 3 thatcan be infected and continue to produce HIV-2. Furthermore,B-lymphoblastic cell lines can also be productively infected by HIV.Montagnier et al, Science, 225:63-66(1984).

The proteins and glycoproteins of the invention can be produced in thetarget cells using the culture conditions previously described, as wellas other standard techniques. For instance, infected human lymphocytescan be stimulated for three days by phytohemaglutinin (PHA). Thelymphocytes can be cultured in RPMI-1640 medium to which has been added10% fetal calf serum, 10⁻⁵ M beta mercaptoethanol, interleukin 2, andhuman alpha anti-interferon serum. Barre-Sinoussi et al, Science,220:868-871 (1983). In addition, techniques for the propagation of HIV-2in HUT 78 and CEM cell lines are described in copending U.S. applicationSer. No. 835,228, filed Mar. 3, 1986, the entire disclosure of which isrelied upon and incorporated by reference herein.

The production of virus in the cell cultures can be monitored usingseveral different techniques. Supernatant fluids in the cell culturescan be monitored for viral reverse transcriptase activity. Electronmicroscopic observation of fixed and sectioned cells can also be used todetect virus. In addition, virus can be detected by transmitting thevirus to fresh normal human T-lymphocytes (e.g., umbilical cord blood,adult peripheral blood, or bone marrow leukocytes) or to establishedT-cell lines. Testing for antigen expression by indirectimmunofluorescence or Western Blot procedures using serum fromseropositive donors can also be employed. In addition, nucleic acidprobes can be utilized to detect viral production.

After a sufficient period of time for viral multiplication to takeplace, infected cells can be separated from the culture medium anddisrupted to expose intracellular proteins using conventionaltechniques. For example, physical sheering, homogenization, sonication,detergent solublization, or freeze-thawing can be employed. The viralproteins released by these cells can be separated from the othercellular components and purified using standard biochemical procedures.For example, proteins can be separated from the live virus bycentrifugation, and the proteins can then be purified byultracentrifugation, gel filtration, ion-exchange chromatography,affinity chromatography, dialysis, or by the use of monoclonalantibodies or by combinations of these procedures. A thoroughpurification of the antigens of the invention can be performed byimmunoreaction with the sera of patients known to possess antibodieseffective against the antigens, with concentrated antibody preparationssuch as polyclonal antibodies, or with monoclonal antibodies directedagainst the antigens of the invention.

The proteins and the glycoproteins of the present invention can be usedas antigens to identify antibodies to HIV-2 and SIV in materials and todetermine the concentration of the antibodies in those materials. Thus,the antigens can be used for qualitative or quantitative determinationof the retrovirus in a material. Such materials of course include humantissue and human cells, as well as biological fluids, such as human bodyfluids, including human sera. When used as a reagent in an immunoassayfor determining the presence or concentration of the antibodies toHIV-2, the antigens of the present invention provide an assay that isconvenient, rapid, sensitive, and specific.

More particularly, the antigens of the invention can be employed for thedetection of HIV-2 by means of immunoassays that are well known for usein detecting or quantifying humoral components in fluids. Thus,antigen-antibody interactions can be directly observed or determined bysecondary reactions, such as precipitation or agglutination. Inaddition, immunoelectrophoresis techniques can also be employed. Forexample, the classic combination of electrophoresis in agar followed byreaction with anti-serum can be utilized, as well as two-dimensionalelectrophoresis, rocket electrophoresis, and immunelabeling ofpolyacrylamide gel patterns (Western Blot or immunoblot.) Otherimmunoassays in which the antigens of the present invention can beemployed include, but are not limited to, radioimmunoassay, competitiveimmunoprecipitation assay, enzyme immunoassay, and immunofluorescenceassay. It will be understood that tubidimetric, colorimetric, andnephelometric techniques can be employed. An immunoassay based onWestern Blot technique is preferred.

Immunoassays can be carried out by immobilizing one of theimmunoreagents, either an antigen of the invention or the antibodies tothe antigen, on a carrier surface while retaining immunoreactivity ofthe reagent. The reciprocal immunoreagent can be unlabeled or labeled insuch a manner that immunoreactivity is also retained. These techniquesare especially suitable for use in enzyme immunoassays, such as enzymelinked immunosorbent assay (ELISA) and competitive inhibition enzymeimmunoassay (CIEIA).

When either the antigen of the invention or antibody to the antigen isattached to a solid support, the support is usually a glass or plasticmaterial. Plastic materials molded in the form of plates, tubes, beads,or disks are preferred. Examples of suitable plastic materials arepolystyrene and polyvinyl chloride. If the immunoreagent does notreadily bind to the solid support, a carrier material can be interposedbetween the reagent and the support. Examples of suitable carriermaterials are proteins, such as bovine serum albumin, or chemicalreagents, such as gluteraldehyde or urea. Coating of the solid phase canbe carried out using conventional techniques.

Depending on the use to be made of the proteins and glycoproteins of theinvention, it may be desirable to label them. Examples of suitablelabels are radioactive labels, enzymatic labels, fluorescent labels,chemiluminescent labels, and chromophores. The methods for labelingproteins and glycoproteins of the invention do not differ in essencefrom those widely used for labeling immunoglobulin. The need to labelmay be avoided by using labeled antibody to the antigen of the inventionor anti-immunoglobulin to the antibodies to the antigen as an indirectmarker.

Once the proteins and glycoproteins of the invention have been obtained,they can be used to produce polyclonal and monoclonal antibodiesreactive therewith. Thus, a protein or glycoprotein of the invention canbe used to immunize an animal host by techniques known in the art. Suchtechniques usually involve inoculation, but they may involve other modesof administration. A sufficient amount of the protein or theglycoprotein is administered to create an immunogenic response in theanimal host. Any host that produces antibodies to the antigen of theinvention can be used. Once the animal has been immunized and sufficienttime has passed for it to begin producing antibodies to the antigen,polyclonal antibodies can be recovered. The general method comprisesremoving blood from the animal and separating the serum from the blood.The serum, which contains antibodies to the antigen, can be used as anantiserum to the antigen. Alternatively, the antibodies can be recoveredfrom the serum. Affinity purification is a preferred technique forrecovering purified polyclonal antibodies to the antigen, from theserum.

Monoclonal antibodies to the antigens of the invention can also beprepared. One method for producing monoclonal antibodies reactive withthe antigens comprises the steps of immunizing a host with the antigen;recovering antibody-producing cells from the spleen of the host; fusingthe antibody-producing cells with myeloma cells deficient in the enzymehypoxanthine-guanine phosphoribosyl transferase to form hybridomas;selecting at least one of the hybridomas by growth in a mediumcomprising hypoxanthine, aminopterin, and thymidine; identifying atleast one of the hybridomas that produces an antibody to the antigen;culturing the identified hybridoma to produce antibody in a recoverablequantity; and recovering the antibodies produced by the culturedhybridoma.

These polyclonal or monoclonal antibodies can be used in a variety ofapplications. Among these is the neutralization of corresopndingproteins. They can also be used to detect viral antigens in biologicalpreparations or in purifying corresponding proteins, glycoproteins, ormixtures thereof, for example when used in affinity chromatographiccolumns.

The invention provides immunogenic proteins and glycoproteins, and moreparticularly, protective polypeptides for use in the preparation ofvaccine compositions against HIV-2. These polypeptides can thus beemployed as viral vaccines by administering the polypeptides to a mammalsusceptible to HIV-2 infection. Conventional modes of administration canbe employed. For example, administration can be carried out by oral,respiratory, or parenteral routes. Intradermal, subcutaneous, andintramuscular routes of adminstration are preferred when the vaccine isadministered parenterally.

The ability of the proteins, glycoproteins, and vaccines of theinvention to induce protective levels of neutralizing antibody in a hostcan be enhanced by emulsification with an adjuvant, incorporation in aliposome, coupling to a suitable carrier, or by combinations of thesetechniques. For example, the proteins and glycoproteins of the inventioncan be administered with a conventional adjuvant, such as aluminumphosphate and aluminum hydroxide gel, in an amount sufficient topotentiate humoral or cell-mediated immune response in the host.Similarly, the polypeptides can be bound to lipid membranes orincorporated in lipid membranes to form liposomes. The use ofnonpyrogenic lipids free of nucleic acids and other extraneous mattercan be employed for this purpose.

The immunization schedule will depend upon several factors, such as thesusceptibility of the host to infection and the age of the host. Asingle dose of the vaccine of the invention can be administered to thehost or a primary course of immunization can be followed in whichseveral doses at intervals of time are administered. Subsequent dosesused as boosters can be administered as needed following the primarycourse.

The proteins and vaccines of the invention can be administered to thehost in an amount sufficient to prevent or inhibit HIV-2 infection orreplication in vivo. In any event, the amount administered should be atleast sufficient to protect the host against substantialimmunosuppression, even though HIV infection may not be entirelyprevented. An immunogenic response can be obtained by administering theproteins or glycoproteins of the invention to the host in an amount ofabout 10 to about 500 micrograms antigen per kilogram of body weight,preferably about 50 to about 100 micrograms antigen per kilogram of bodyweight. The proteins and vaccines of the invention can be administeredtogether with a physiologically acceptable carrier. For example, adiluent, such as water or a saline solution, can be employed.

In summary, proteins and glycoproteins, which are precursors of HIV-2and SIV envelope protein, have now been identifed. In addition toproviding useful tools for detection of antibodies to the retrovirus inhumans and for raising neutralizing antibodies to HIV-2 in vitro and invivo, this invention adds to the base of knowledge relating toimmunodeficiency active proteins and glycoproteins of the AIDS viruses.

The reference molecular weight was in the form of the following dyemarkers marketed by BRL Co.:

myosine 200 Kd

phosphorylase B 92.7 Kd

BSA 68 Kd .

ovalbumin 43 Kd

alpha chymotrypsin 25.7 Kd.

beta lactoglobulin 18.4 Kd.

lysozyme 14.3 Kd.

Molecular weights were estimated within an accuracy of about ±10%.

REFERENCES

Anderson, R. G. W. and Pathat, R. K. (1985). Vesicles and cisternae inthe Trans Golgi apparatus of human fibroblasts are acidic compartments.Cell 40, 635-643.

Barre-Sinoussi, F. Chermann, J. C., Rey, F., Nugeyre, M. T., Chamaret,S., Gruest, J., Dauguet, C., Axler-Blin, C., Vezinet-Brun, F., Rouzioux,C., Rozembaum, W. and Montagnier, L., (1983) : Isolation of aT-lymphotropic retrovirus from a patient at risk for acquired immunedeficiency syndrome (AIDS). Science 220, 868-871.

Berg, K., (1977). Sequential antibody affinity chromatography of humanleucocyte interferon. Scand. J. Immunol. 6, 77-86.

Brun-Vezinet, F., Rey, M. A., Katlama, C., Girard, P. H., Roulot, D.,Yeni, P., Lenoble, L., Clavel, F., Alizon, M., Gadelle, S., Madjar, J.J. and Harzic, M., (1987). Lymphadenopathy-associatied virus type 2 inAIDS and AIDS-realted complex. Lancet i: 128-132.

Chakrabarti, L., Guyader, M., Alizon, M., Daniel, M. D., Desrosiers, R.C., Thiollais, P. and Sonigo, P. (1987). Sequence of simianimmunodeficiency virus from macaque and its relationship to other humanand simian retroviruses. Nature 328, 543-547.

Clavel, F., Guetard, D., Brun-Vezinet, F., Chamaret, S., Rey, M. A.,Santos-Ferreira, M. O., Laurent, A. G., Dauguet, C., Katlama, C.,Rouzioux, C., Klatzmann, D., Champalimaud, J. L., and Montagnier, L.(1986a). Isolation of a new retrovirus from West African patients withAIDS. Science 233, 343-346.

Clavel, F., Guyader, M., Guetard, D., Salle, M., Montagnier, L. andAlizon, M. (1986b). Molecular cloning and polymorphism of the immunedeficiency virus type 2. Nature 324: 691-694.

Clavel, F., Mansinho, K., Charet, S., Guetard, D., Favier, V., Nina, J.,Santos-Ferreira, M. O., Champalimaud, J. L., and Montagnier, L. (1987).Human immunodeficiency virus type 2 infection associated with AIDS inWest Africa. N. Eng. J.Med. 316, 1180-1185.

Dalgleish, A. G., Beverley, P. C., Clapham, P. R., Crawford, D. H.,Greaves, M. F. and Weiss, R. A. (1984). The CD4 (T4) antigen is anessential component of the receptor for the AIDS retrovirus. Nature,312, 763-767.

Daniel, M. D., Letvin, N. L., King, N. W., Kannagi, M., Sehgal, P. Hunt,R. D., Kanki, P. J., Essex, M. and Desrosiers, R. C. (1985) Isolation ofa T-cell tropic HTLV-III-like retrovirus from macaques. Science 228,1201-1204.

Datema, R., Romero, P. A., Legler, G. and Schwartz, R. T. (1982)Inhibition of formation of complex oligosaccharides by the glucosidaseinhibitor bromoconduritol. Proc. Natl. Acad. Sci. USA 79, 6787-6791.

Franchini, G., Gurgo, C., Guo, H-G., Gallo, R. C., Collalti, F.Fargnoli, K. A., Hall, L. F., Wong-Staal, F. and Reitz Jr, M. S. (1987).Sequence of simian immunodeficiency virus and its relationship to thehuman immunodeficiency viruses. Nature 328, 539-543.

Fuhrmann, U., Bause, E., Legler, G. and Ploegh, H. (1984). Novelmonnosidase inhibitor blocking conversion of high mannose to complexoligosaccharides. Nature 307, 755-758.

Fuhrmann, U., Bause, E. and Ploegh, H. (1985). Inhibitors ofoligosaccharide processing. Biochimica et Biophysica Acta 825, 95-110.

Fultz, P. N., McClure, H. M., Anderson, D. C., Swenson, R. B., Anand, R.and Srinivasan, A., (1986). Isolation of a T-lymphotropic retrovirusfrom naturally infected sooty mangabey monkeys (Cercocebusatys). Proc.Natl. Acad. Sci. 83, 5286-5290.

Gallaher, W. R. (1986). Detection of a fusion peptide sequence in thetransmembrane protein of human immunodeficiency virus. Cell 50, 327-328.

Gazdar, A. F., Carney, D. N., Bunn, P. A., Russel, E. K., Jaffe, E. S.,Schechter, G. P. and Guccion, J. G. (1980). Mitogen requirements for thein vitro production of cutaneous T-cell lymphomas. Blood 55, 409-417.

Griffiths, G., Quinn, P. and Warren G. (1983). Dissection of Golgicomplex. I. Monensin inhibits the transport of viral membrane proteinsfrom medial to trans Golgi cisternae in baby hamster kidney cellsinfected with Semliki Forest virus. J. Cell. Biol. 96, 835-851.

Griffiths, G. and Simons, R. (1986). The trans Golgi network: sorting atthe exit site of the Golgi complex. Science 234, 438-443.

Guyader, M., Emerman, M., Sonigo, P., Clavel, F., Montagnier, L. andAlizon, M. (1987). Genome organization and transactivation of the humanimmunodeficiency virus type 2. Nature 326: 662-669.

Heifetz, A., Keenan, R. W. and Elhein, A. D. (1979). Mechanism of actionof tunicamycin on the UDP-GlcNAc: dolichylphosphate GlcNAc-1-phosphatetransferase. Biochemistry 18, 2186-2192.

Johnson, D. C. and Schlesinger M. J. (1980). Vesicular stomatitis virusand sindbis virus glycoprotein transport to cell surface is inhibited byionophores. Virology 103, 407-424.

Kannagi, M., Yetz, J. M., and Letvin, N. L. (1985). In vitro growthcharacteristics of simian T-lymphotropic virus type III Proc. Natl.Acad. Sci. USA 82, 7053-7057.

Klatzmann, D., Champagne, E., Chamaret, S., Gruest, J. Guetard, D.,Hercend, T., Gluckman, J. C. and Montagnier, L. (1984). T.lymphocyte T4molecule behaves as the receptor for human retrovirus LAV. Nature 312,767-768.

Kornfeld, R. and Kornfeld, S. (1985). Assembly of asparagine-linkedoligosaccharides. Ann. Rev. Biochem. 54, 631-664.

Kowaslski, M., Potz, J., Basiripour, L., Dorfman, T., Goh, W. C.Terwilliger, E., Dayton, A., Rosen, C., Haseltine, W. and Sodroski,(1987). Functional regions of the envelope glycoprotein of humanimmunodeficiency virus type 1. Science 237, 1351-1355.

Krust, B., Laurent, A. G., Le Guern, A., Jeannequin, O., Montagnier, L.and Hovanessian, A. G. (1988). Characterization of a monoclonal antibodyspecific for HIV-1 precursor glycoprotein. AIDS 2, 17-24.

Lemansky, P., Gieselmann, V., Hasilik, A. and Von Fugura, K. (1984).Cathespsin D and β-hexosaminidase synthesized in the presence of1-deoxynojirimycin accumulate in the endoplasmic reticulum. J. Biol.Chem. 259, 10129-10135.

Levy, J. A., Hoffman, A. D., Kramer, S. M., Lanois, J. A., Shimabukuro,J. M. and Oskiro, L. S. (1984). Isolation of lymphocytopathicretroviruses from San Francisco patients with AIDS. Science 225,840-842.

Li, E., Tabas, I. and Kornfeld, S.(1978). The synthesis of complex-typeoligosaccharides. I. Structure of the lipid-linked oligosaccharide ofthe vesicular stomatitis virus G protein. J. Biol. Chem. 253, 7762-7770.

Lifson, J. D., Feinberg, M. B., Reyes, G. R., Rabin, L., Banapour B,Chakrabati, S., Moss, B., Wong-Staal, F., Steimer,, K. S. and Engleman,E. G. (1986). Induction of CD4-dependent cell fusion by the HTLV-III/LAVenvelope glycoprotein. Nature 323, 725-728.

Lodish, H. F. and Kong, N. (1984). Glucose removal from N-linkedoligosaccharides is required for efficient maturation of certainsecretory glycoproteins from the rough endoplasmic reticulum to theGolgi complex. J. Cell. Biol. 98, 1720-1729.

Marsh, M. and Dalgleish, A. (1988). How do human immunodeficiencyviruses enter cells ? Immunology Today 8, 369-371.

McClure, M. O., Marsh, M. and Weiss, R. A. (1988). Humanimmunodeficiency virus infection of CD4-bearing cells occurs by apH-independent mechanism. EMBO J. 7, 513-518.

McDougal, J. S., Mawle, A., Cort, S. P., Nicholson, J. K. A., Cross, G.D., Scheppler-Campbel, J. A., Hicks, D. and Sligh J. (1985). Cellulartropism of the human retrovirus HTLV-III/LA I. Role of T cell activationand expression of the T4 antigen. J. Immunol. 135, 3151-3162.

McDougal, J. S., Kennedy, M. S., Sligh, J. M. Cort, S. P., Mawle, A. andNicholson, J. K. A. (1986). Binding of HTLV-III/LAV to T4+ cells by acomplex of the 110K viral protein and the T4 molecule. Science 231,382-385.

Montagnier, L. and Alizon M. (1987). The human immune deficiency virus(HIV): an update. Ann. Inst. Pasteur/Virol. 138, 3-11

Montagnier, L., Chermann, J. C., Barre-Sinoussi, F., Chamaret, S.Gruest, J., Nugeyre, M. T., Rey, F., Dauguet, C., Axler-Blin, C.Vezinet-Brun, F., Rouzioux, C., Saimot, A. G., Rozembaum, W., Gluckman,J. C., Klatzmann, D., Vilmer, E., Griscelli, C., Gazengel, C. andBrunet, J. B. (1984). A new human T-lymphotropic retrovirus :characterization and possible role in lymphadenopathy and acquiredimmune deficiency syndrome. In human T cell leukemia/lymphoma viruses,edited by Gallo, R. C. Essex, M. E., Gross, L. Cold Spring HarborLaboratory, New York, 1984, pp 363-379.

Montagnier, L., Clavel, F., Krust, B., Chamaret, S., Rey, F.,Barre-Sinoussi, F., and Chermann, J. C. (1985). Identification andantigenecity of the major envelope glycoprotein of lymphadenopathyAssociated Virus. Virology 144, 283-289.

Novikoff, A. B. (1976). The endoplasmic reticulum: a cytochemist's view:Proc. Natl. Acad. Sci. USA 73, 2781-2787

O'Farrel, P. H. (1975). High resolution two-dimensional electrophoresisof proteins. J. Biol. Chem. 250, 4007-4021.

Olden, K, Pratt, R. M. and Yamada, K. M. (1978). Role of carbohydratesin protein secretion and turnover: effects of tunicamycin on the majorcell surface glycoprotein of chick embryo fibroblasts. Cell 13, 461-473.

Orci, L., Ravazzola, M. Amherdt, A. P., Powel, S. K., Quinn, D. andMoore, H-P. H. (1987). The trans-most cisternae of the Golgi complex: acompartment for sorting of secretory and plasma membrane proteins. Cell51, 1039-1051.

Peyrieras, N., Bause, E., Legler, G., Vasilof, R. Claesson, L. Peterson,P. and Ploegh, H. (1983). Effects of glucosidase inhibitors nojirimycinand deoxynojirimycin on the biosynthesis of membrane and secretoryglycoproteins. EMBO J. 2, 823-832.

Sonigo, P., Alizon, M., Staskus, K., Klatzmann, D., Cole, S., Danos, O.,Retzel, E. Tiollais, P., Haase, A. and Wain-Hobson, S. (1985).Nucleotide Sequence of the visna lentivirus: relationship to the AIDSvirus. Cell 42, 369-382.

Stein, B. S., Gowda, S. D., Lifson, J. D., Penhallow, R. C., Bensch, K.G., and Engleman, E. G. (1987). pH-independent HIV-entry intoCD4-positive T cells via virus envelope fusion to plasma membrane. Cell49, 659-668.

Strous, G. J. A. M. and Lodish, H. F. (1980). Intracellular transport ofsecretory and membrane proteins in hepatoma cells infected by vesicularstomatitis virus. Cell 22, 709-717.

Tarentino, A. L., Plummer, T. H. and Maley, F. (1974). The release ofintact oligosaccharides from specific glycoproteins by endo-B-N-acetylglucosaminisase H. J. Biol. Chem 249, 818-824.

Tartakoff, A. M. and Vassalli, P. (1977). Plasma cell immunoglobulinsecretion: arrest is accompanied by alterations of the Golgi complex. J.Exp. Med. 146, 1332-1345.

Wain-Hobson, S., Sonigo, P., Danos, O., Cole, S., and Alizon, M.(1985a). Nucleotide sequence of the AIDS virus LAV. Cell 40, 9-17.

Wain-Hobson, S., Alizon, M., and Montagnier, L. (1985b). Relationship ofAIDS to other retroviruses. Nature 313, 743.

Weiss, R. A. (1988). Receptor molecule block HIV. Nature 331,15.

What is claimed is:
 1. A glycoprotein of human immunodeficiency virustype 2 (HIV-2) wherein(A) said glycoprotein is a precursor of envelopeprotein of HIV-2; (B) said glycoprotein has an apparent molecular weightof about 300 kD (gp300); and (C) said glycoprotein is isolated fromother HIV-2 proteins and glycoproteins.
 2. A protein of humanimmunodeficiency virus type 2 (HIV-2), wherein(A) said protein is aprecursor of an envelope protein of HIV-2; (B) said protein has anapparent molecular weight of about 200 kD (p200); (C) said protein issubstantially unglycosylated; and (D) said protein is isolated fromother HIV-2 proteins and glycoproteins.
 3. A protein of humanimmunodeficiency virus type 2 (HIV-2), wherein(A) said protein is aprecursor of an envelope protein of HIV-2; (B) said protein has anapparent molecular weight of about 90 to about 80 kD (p90/80); (C) saidprotein is substantially unglycosylated; and (D) said protein isisolated from other HIV-2 proteins and glycoproteins.
 4. A glycoproteinof simian immunodeficiency virus (SIV), wherein(A) said glycoprotein isa precursor of an envelope glycoprotein of SIV; (B) said glycoproteinhas an apparent molecular weight of about 300 kD (gp300_(SIV)); and (C)said glycoprotein is isolated from other SIV proteins and glycoproteins.5. A labeled antigen comprising a glycoprotein of human immunodeficiencyvirus type 2 (HIV-2), wherein(A) said glycoprotein is a precursor ofenvelope protein of HIV-2; (B) said glycoprotein has an apparentmolecular weight of about 300 kD (gp300); (C) said glycoprotein isisolated from other HIV-2 proteins and glycoproteins; and (D) saidantigen is labeled with an immunoassay label selected from the groupconsisting of radioisotope, enzyme, fluorescent, chemiluminescent, andchromophore labels.
 6. A labeled antigen comprising a glycoprotein ofsimian immunodeficiency virus (SIV), wherein(A) said glycoprotein is aprecursor of envelope protein of SIV; (B) said glycoprotein has anapparent molecular weight of about 300 kD (gp300_(SIV)); (C) saidglycoprotein is isolated from other SIV proteins and glycoproteins; and(D) said antigen is labeled with an immunoassay label selected from thegroup consisting of radioisotope, enzyme, fluorescent, chemiluminescent,and chromophore labels.